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A novel form of RNA double helix based on G·U and ·A+ wobble base pairing

ANKUR GARG1,2 and UDO HEINEMANN1,2 1Crystallography, Max-Delbrück Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany 2Institute for Chemistry and , Freie University Berlin, 14195 Berlin, Germany

ABSTRACT Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double- helical structures of nucleic . We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs ( helix) with two G·U and four rare C·A+ wobble pairs. Four within the W helix are N1-protonated and wobble-base-paired with the opposing through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson–Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA and pH . Keywords: RNA double helix; wobble base pairing; wobble helix; N1 protonation; pH-dependent

INTRODUCTION angles to form two stable hydrogen bonds (Hunter et al. 1987; Puglisi et al. 1990). The formation of two isosteric hy- Base-pairing in most nucleic acids follows the Watson–Crick drogen bonds in a C·A wobble pair, however, is not possible pairing rules. Non-Watson–Crick base-pairing, however, is with bases in their standard configuration. Here, the N1(A)– also prevalent and plays crucial roles for various physiological O2(C) can only form if either the proton- functions (Deng and Sundaralingam 2000; Masquida and ation or the tautomeric state of the participating bases are Westhof 2000; Varani and McClain 2000). Among all non- changed. A first model proposes hydrogen-bond stabilization Watson–Crick pairs, the G·U wobble is the most studied by rare amino–imino tautomers of cytosine or adenine pair that has been shown to be highly conserved in the accep- (Saenger 1983; Hunter et al. 1986, 1987; Russo et al. 1998; tor helix of tRNAAla, common in other tRNAs (Sprinzl et al. Masoodi et al. 2016). Similarly, a second model suggests pro- 1998) and rRNA (Gautheret et al. 1995), and critical tonation of cytosine N3 simultaneously with a tautomeric for RNA– recognition (Hou and Schimmel 1988; shift in the adenine base (Supplemental Fig. S1). Cytosine McClain and Foss 1988) and splice-site selection in group I N3 protonation has been invoked in U6 RNA loop structure (Cech 1987; Strobel and Cech 1995). Among other formation in Trypanosoma brucei and Crithidia fasciculata mismatches, C·A+ wobble pairs are less frequently observed; (Huppler et al. 2002) and the DNA–triostin-A interaction however, they have been shown to be isosteric with and capa- (Quigley et al. 1986). However, whereas the transient forma- ble of substituting G·U pairs in some cases (Samuelsson et al. tion of base pairs involving the rare tautomers of 1983; Doudna et al. 1989; Gautheret et al. 1995; Masquida and was recently demonstrated by NMR and Westhof 2000). methods (Kimsey et al. 2015), experimental proof of C·A G·U and C·A+ pairs are significantly different from Watson– pairs with adenine imino tautomers is lacking, to the best Crick base pairs, and they present opportunities for specific of our knowledge. recognition either by generating local irregularity of helical The most frequently invoked model for C·A mismatch structure or by introducing electrostatic variations in the formation proposes protonation of adenine N1, which would grooves. G·U wobble base pairs are thermodynamically fa- vorable and require only slight adjustment in λ © 2018 Garg and Heinemann This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see Corresponding author: [email protected] http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is avail- Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.064048. able under a Creative Commons License (Attribution-NonCommercial 4.0 117. International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Garg and Heinemann allow a second hydrogen bond to form with cytosine O2 as tioned a partial RNA model into the unit with log- acceptor atom (Hunter et al. 1986, 1987). In aqueous solu- likelihood gain (LLG) and function -score tion, exists in three different mono-protonation (TFZ) of 54.4 and 5.3, respectively. One round of automated states with pKa values of 3.64, −1.53, and −4.02 for N1, model building and refinement was performed using N7, and N3 protonation (Saenger 1983; Kapinos et al. Autobuild wizard, which, after fitting the correct oligonucle- 2011), and the protonation propensity of adenosine N1 was otide sequence and refinement, yielded Rwork/Rfree of 23.7%/ reported as being 96.1% over N7 and N3 protonation states 24.0%. Bond and angle restraints combined with base- (Kapinos et al. 2011). Also, adenine N1 protonation has planarity and hydrogen-bonding restraints for base pairs been observed in poly(rA) fibers (Rich et al. 1961), oligo were used simultaneously with anisotropic displacement (rA) stretches in crystal structures (Gleghorn et al. 2016), factor restraints over the whole refinement process, which and an NMR structure of poly(dA) (Chakraborty et al. yielded a final Rwork/Rfree of 12.9%/15.1% (Table 1). 2009), allowing the oligo(A) to form either a parallel-strand- The RNA molecule crystallized in space group P6322, ed double helix (π-helix) at acidic pH or a single-stranded forming an 18-bp antiparallel RNA double helix with two helical structure at neutral pH. identical strands related by crystallographic symmetry. In addition, the presence of adenine N1 protonation in These duplexes are stacked in head-to-tail fashion to generate + isolated or tandem C·A wobble base pairs is confirmed by a pseudo-continuous RNA column around the 63 screw axis several crystal (Hunter et al. 1986, 1987; Jang et al. 1998; along the crystallographic c-axis (Supplemental Fig. S2A). Pan et al. 1998) and NMR (Puglisi et al. 1990; Durant and The helical stacks are stabilized by eight well-coordinated Davis 1999; Huppler et al. 2002) structures of different oligo- molecules present in the minor groove at the helical . Interestingly, tandem C·A+/A+·C pairs exhibit junction. The eight are symmetrically arranged and cross-strand stacking due to the near 0° helical twist, form a hydrogen-bonding network that stabilizes the helical which is compensated by a twist increase by 10°–15° at the arrangement by forming specific hydrogen bonds with all adjacent Watson–Crick pairs (Jang et al. 1998), while an iso- four 2′OH, N3, and O2 atoms of adenine and , lated C·A+ pair causes little helical irregularity, suggesting respectively (Supplemental Fig. S2B). that neighboring base pairs are efficient in maintaining over- all helical geometry when more than one C·A+ pair is present. Geometry of RNA double helix Although several structures for base pair mismatches are available showing G·U or C·A+ wobble pairs in isolation, in In the crystal, two RNA strands form an antiparallel double tandem or with other mismatches, both wobble pairs have helical structure with 18 bp (Fig. 1). The helical parameters never been shown together in a crystal structure. Only one for the full-length RNA structure (fl helix) suggest that the NMR structure of an RNA hairpin with both G·U and double helix exhibits A-RNA conformation with helical C·A+ pairs is available that shows these wobble pairs in isola- periodicity of 10.96 bp, similar to canonical A-RNA, which tion (Puglisi et al. 1990). has 11 bp per helical turn (Table 2; Schindelin et al. 1995; Here we present a high-resolution crystal structure of an Olson et al. 2001). The RNA double helix can be divided 18-bp antiparallel RNA double helix, which includes one into three helical segments of 6 bp each with the first and half helical turn of W helix, comprising six contiguous wob- third segments (WC helix) having a total of 12 bases paired ble base pairs at the center flanked by half turns of standard A-form RNA on either side. The W-helical stretch is formed by four C·A+ and two G·U wobble base pairs. The geometry TABLE 1. -ray data collection and refinement statistics of the novel form of RNA helix is described in detail with a Data collection statistics focus on the central wobble pairs. In addition, pH-dependent Space group P6322 variations in the RNA structure are probed by various a, , c (Å) 45.00, 45.00, 94.20 Resolution (Å) 38.97–1.38 (1.46–1.38) biophysical methods. We suggest that the pH-dependent meas (%) 9.4 (162.3) formation of W-helical RNA might become a valuable tool 15.08 (1.50) for RNA nanotechnology applications. CC1/2 1.0 (0.69) Completeness (%) 99.7 (99.8) Multiplicity 10.47 RESULTS Refinement statistics Resolution (Å) 38.97–1.38 (1.46–1.38) Structure analysis and crystal packing No. of reflections 12,286 R R X-ray diffraction data for the RNA, r(UGUUCUCUACGAA work/ free 0.129/0.151 Average B-factors (A2) GAACA), were collected to maximum resolution of 1.38 Å, RNA (nonhydrogen) 17.30 and a Matthews’ analysis indicated the presence of one Water 34.90 RNA strand in the asymmetric unit. Molecular replacement with PHASER-MR (McCoy et al. 2005) oriented and posi- Values in parentheses represent the highest resolution shell.

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A novel RNA helix based on wobble base pairing

(Hunter et al. 1986, 1987; Pan et al. 1998) or in tandem (Biswas et al. 1997; Jang et al. 1998) has been reported earlier. The structure presented here shows that up to six contiguous non-Watson–Crick base pairs may be accommodated in double-helical RNA. All in the are in C3′-endo pucker with average pseudorotation phase angle of 13.10 (±3.92)°, and all are in anti conformation having an average glyosidic torsion angle of −162.76 (±5.64)°. Most back- bone torsion angles are within the expected regions for the A conformation, having gauche (−) for α and ζ, trans for β and ε, and gauche (+) for γ and δ, with the exception of the C5 which has both α and γ torsions in trans with 143.8° and −169.7°, respectively. Although these trans–trans conformations do not occur at every single wobble residue, they occur frequently in structures with wobble G·U or C·A+ pairs (Biswas et al. 1997; Jang et al. 1998; Pan et al. FIGURE 1. X-ray crystal structure of antiparallel RNA double helix 1998). with 2DFo-mFc difference density contoured at 1.0 σ with 1.6 Å radius of atoms. (A) The crystallographic asymmetric unit consists of a single 18-nt RNA strand. (B) Crystallographic dyad symmetry generates an RNA helix is stabilized by 4 C·A+ and 2 G·U wobble RNA double helix (fl helix) that is divided into three segments of 6 base pairs bp each, abbreviated as WC, W, and WC helix. In the WC-helix seg- ments, bases are paired in Watson–Crick geometry, while the WB helix Antiparallel arrangement of r(UGUUCUCUACGAAGAACA) contains six bases paired in wobble geometry. strands is supported by a crystallographic dyad axis perpen- dicular to the helix axis that passes at the A9-C10/A9-C10 according to canonical Watson–Crick base-pairing rules, base pair step and generates nine unique base pairs at either while the second helical segment (W helix) at the center con- end of the helix. Six terminal base pairs forming the WC helix tains six bases per strand paired in a noncanonical scheme are stabilized by Watson–Crick base-pairing with A·U and (see Fig. 1). Helical parameters for the WC helix reflect A- C·G pairs, respectively (Supplemental Fig. S3). The striking RNA-like geometry with periodicity of 10.92 bp per turn; feature of the RNA helix is the presence of six non- however, the W helix displays unique features with similari- Watson–Crick base pairs at the center (W helix) with three ties to both A-RNA and A′-RNA. Although large local varia- of them (C10·9A+, G11·8U, A+12·7C) being crystallographi- tions in helical Twist and Rise are present, the mean values of cally unique pairs. All three unique wobble pairs are depicted 29.09° for the helical Twist and 1.91 Å for the Rise, respective- in Figure 2 with their water molecule coordination. All six ly, reflect similarities between the W helix and A-RNA, while non-Watson–Crick base pairs display clear wobble geometry, the W helix is similar to A′-RNA in having helical periodicity which is characterized by opposite drifts of λ angles for of 12 bp (Supplemental Table S1; Arnott et al. 1972; Tanaka and and formation of two hydrogen et al. 1999). This indicates that locally distorted wobble base bonds, one of which links the purine imino hydrogen and pair repeats can be incorporated in RNA double helices with the carbonyl oxygen. little overall distortion in helical geometry. Incorporation In the W helix, all direct interbase hydrogen bonds are of maximally four noncanonical base pairs (Holbrook et al. facilitated by the reduction in the C1′–C1′ distance to an av- 1991) or two wobble-base pairs present either in isolation erage of 10.3 Å from 10.6 Å, as observed in Watson–Crick

TABLE 2. Mean values for global helical parameters of the RNA fl helix, WC helix, and W helix

Structure Twist (°) Rise (Å) Inclination (°) X-displacement (Å)

fl helix 32.83 (7.94) 2.52 (0.73) 17.73 (11.82) −5.14 (3.87) WC helix 32.96 (2.53) 2.69 (0.17) 16.90 (5.71) −4.34 (1.15) W helix 29.09 (13.57) 1.91 (1.14) 23.87 (19.57) −7.96 (6.41) A RNA(1SDR) 33.36 (3.54) 2.69 (0.42) 16.79 (7.26) −4.42 (1.27) A DNA 32.50 (3.80) 2.83 (0.36) 14.70 (7.30) −4.17 (1.22) B DNA 36.50 (6.6) 3.29 (0.21) 2.1 (9.2) 0.05 (1.28)

Values for standard A-DNA and B-DNA and an A-RNA dodecamer structure are given for comparison. Standard deviations are given in parentheses.

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FIGURE 2. 2DFo-mFc electron density contoured at 1.0 σ for the three unique wobble base pairs in the WB helix. Two characteristic hy- drogen bonds are formed between bases, and a third hydrogen bond is mediated by a water molecule located in the major groove. (A,C) Two unique C·A+ pairs and (B) the unique G·U pair shown with their hydration networks in both helical grooves. () The C·A base pair after refinement with relaxed geometric restraints. Characteristic bond-angle changes in the adenine and positive difference density (green) at the position of adenine H1 both indicate adenine N1 protonation. The Fo–Fc difference density is contoured at the 2.5 σ level. Hydrogen-bond distances are given in Ångstrom units with hydrogen-acceptor distances shown in black and donor-acceptor distances (… or O…O) in cyan. pairs (Rosenberg et al. 1976; Seeman et al. 1976; Olson et al. each other. Major- and minor-groove water molecules, as 2001). Simultaneously, the bases of a wobble pair undergo a observed near the C·A+ base pairs, and minor-groove waters, shear movement, leading to an average −12.5° and +15° shift as bound to the G·U base pair, present either in isolation or in in λ angle for the purine and pyrimidine base, respectively tandem, have been shown to be instrumental in stabilizing (Supplemental Figure S4; Supplemental Table S2; Hunter the wobble pair and its incorporation in RNA helices et al. 1986; Pan et al. 1998). Both G·U pairs are organized (Hunter et al. 1987; Holbrook et al. 1991; Biswas and in well-defined wobble geometry with two hydrogen bonds Sundaralingam 1997; Biswas et al. 1997; Pan et al. 1998; between O6(G)…N3(U)- and N1(G)-H…O2(U). In Mueller et al. 1999; Trikha et al. 1999). The novel feature C·A+ pairs, one standard hydrogen bond is formed between of the structure presented here lies in the demonstration N3(C)…N6(A)-H, and a second hydrogen bond is formed that extended segments of exclusively wobble-base-paired between O2(C)…N1(A+)-H to complete the wobble ar- nucleotides can be accommodated in an RNA double helix rangement. The protonation of adenine N1 (see below), facil- with almost no difference in the hydration pattern of the in- itating formation of the second H bond is probably linked to dividual bases. the acidic pH 4.0 of the buffer, which is close to To determine conformational features of the fl helix and the pKa of adenine N1 (Saenger et al. 1975; Saenger 1983; the central W helix, we analyzed the RNA structure with Kapinos et al. 2011). 3DNA (Supplemental Table S3; Zheng et al. 2009). The The unique wobble base pairs C10·9A+ and A+12·7C Shear parameter is generally assumed to be a characteristic exhibit similar hydrogen bonding patterns as G11·8U (see indicator of wobble base pairs because of their approximately Fig. 2). Minor groove water molecules interact with the 2′ ±2 Å Shear which does not occur in Watson–Crick base pairs hydroxyl groups of cytosine, but fail to make any direct hy- (Olson et al. 2001). Notably, in the central W helix, base pairs drogen bond with the paired adenosine. The C·A+ pairs share are significantly distorted with extremes of Shear of ±2.65 Å, with the G·U pair a unique water molecule located in the whereas the flanking WC helix shows nonsheared base pairs major groove, which stabilizes the RNA helix by bridging (Fig. 3A). Additionally, base pairs in the W helix show signif- N4 to N6 of cytosine and adenine, respectively. The icantly higher Opening accompanied by reduced Propeller G11·8U pair is highly hydrated, and its hydrogen bonding distortion, which allows a sufficiently close approach of potential is saturated by an integral water molecule in the hydrogen-bonded atoms (Fig. 3B,C). Similar variations are minor groove, interacting with a free N2 amino group, O2 observed in base-step Twist in the W helix, but the significant and O2′. Unlike the C·A+ base pairs, the G·U pair binds effect of individual base-pair Twist is compensated by under- two water molecules in the major groove, which form a hy- and over-twisting of associated base pairs allowing the RNA drogen bonding network with N7, O6, and O4, and with helix to maintain A configuration (Fig. 3D).

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A novel RNA helix based on wobble base pairing

FIGURE 3. Base pair parameters of the symmetric RNA double helix. Prominent variations in (A) Shear,(B) Opening,(C) Propeller, and (D) Twist steps are observed in the central wobble-base-paired W-helix segment as compared to the terminal Watson–Crick-paired A-RNA.

Wobble helix and adenine protonation poly(A) double helix (Gleghorn et al. 2016). For the crystal structure, this analysis strongly supports the model that the The protonation state of adenine can be inferred by measur- adenine N1 position in the W-helical center is protonated, ing the internal bond angles, and the pattern of bond-angle which facilitates the formation of the O2(C)…N1(A+)-H changes may hint at protonation at a particular in hydrogen bond. Additionally, in the RNA helix the average the adenine ring (Taylor and Kennard 1982; Saenger 1983; C2–N3–C6 bond angle for Watson–Crick paired adenine is Safaee et al. 2013; Gleghorn et al. 2016). After one round of 110.44° (±2.65°), while it moderately increases to 114.52° structure refinement with relaxed geometric restraints, we (±1.19°) in wobble-paired adenines. This increment, howev- compared average C2–N1–C6 and C2–N3–C4 bond angles er, is too small to suggest adenine N3 protonation when com- of adenines in Watson–Crick and wobble base pairs, respec- pared to the CSD or quantum mechanics–based standards of tively, with bond angle standards for different adenine proton- 117.3° (±0.6°) and 116.9°. ation states generated either from 467 small-molecule To rule out the possibility of N3-protonated cytosine-me- structures retrieved from the Cambridge Structural Database diated wobble bond formation (see Supplemental Fig. S1C), (CSD) or computed using quantum mechanics (Table 3; we also compared the C2–N3–C4 bond angles in restrained Gleghorn et al. 2016). Bond angles for individual adenines and unrestrained RNA structures. Upon relaxing the geomet- in the crystal structure are listed in Supplemental Table S4. ric restraints, the average C2–N3–C4 bond angle in wobble Upon relaxing the geometric restraints for RNA, the ade- base pairs did not increase to ∼125° as expected for N3-pro- nine rings in the wobble base pairs readjusted themselves tonated cytosine (Clowney et al. 1996), but instead it reduced in the electron density, resulting in increased C2–N1–C6 to 114.57° (±0.57°) from 118.94° (±0.40°), clearly ruling out bond angles and hydrogen bond lengths. Adenine geometries protonation at cytosine N3. The average C2–N3–C4 bond in Watson–Crick base pairs did not display similar changes. angle in Watson–Crick pairs did not significantly change In wobble base pairs only, low-level difference density ap- upon removing the geometric restraints. peared near the N1 position indicating its protonation (see Fig. 2D). The average C2–N1–C6 bond angle for Watson– Crick paired adenines remained at 117.39° (±0.75°) while pH-dependent UV thermal melting wobble-paired adenines exhibited an increased bond angle of 125.92° (±2.71°). C2–N1–C6 bond-angle values derived To test our hypothesis that formation of the 18-bp RNA from the CSD and quantum mechanics were ∼124.0° (±2.5°) duplex observed in the crystal was facilitated by adenine N1 and 123.4°, respectively, and, similarly, a 126.8° bond angle protonation in its central W-helix segment, we examined if is reported for N1-protonated adenine in a staggered zipper any pH-induced structural transition in the RNA can be

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TABLE 3. Bond angle analysis of adenines present in the WC- and W-helical segments of the crystal structure. Corresponding bond angles for adenine in different protonation states derived from the CSD and quantum mechanics (Gleghorn et al. 2016) are given for comparison.

CSD-derived values (°)

Angle Neutrala N1 protonatedb N3 protonatedc

C2–N1–C6 118.6 ± 1.1 124.0 ± 2.5 119.3 ± 0.8 C2–N3–C4 110.7 ± 1.2 111.7 ± 0.9 117.3 ± 0.6

Quantum chemistry derived (B3LYP) values (°) for adenosine

Angle Neutral N1 protonated N3 protonated N1 and N3 protonated

C2–N1–C6 118.6 123.4 120.1 124.3 C2–N3–C4 111.6 113.8 116.9 117.8

Restrained Unrestrained

Angle Wobble pairs Watson–Crick pairs Wobble pairs Watson–Crick pairs

C2–N1–C6 118.3 ± 0.8 118.3 ± 0.7 126.9 ± 2.7 117.5 ± 1.5 C2–N3–C4 110.9 ± 0.2 110.5 ± 0.9 115.0 ± 0.7 110.5 ± 2.8

a262 adenines. b65 adenines. c12 adenines. observed in thermal melting experiments at different pH val- that the configuration present under physiological conditions ues. UV spectra were recorded at 260 nm and pH values of is thermodynamically favored over the double-helical struc- 3.5, 6.5, and 8.0 to monitor structural transitions upon ture with central W-helix segment observed in crystals grown change from acidic to slightly basic medium. One represen- at low pH. tative out of three experiments is shown in Figure 4A. Upon sample heating, all spectra showed a hyperchromic Differential scanning calorimetry transition in UV absorption, suggesting the dissociation from double-helical to single-stranded RNA. We observed To further determine the thermodynamic properties associ- that pH change from 3.5 to 6.5 raised the RNA melting tem- ated with different conformations observed at different pH perature from ∼55°C to ∼67°C. Notably, no change in melt- values, DSC experiments were performed with RNA ele- ing temperature was observed for a further pH increase from ments buffered at pH 3.5, 6.5, and 7.4. DSC profiles and ther- 6.5 to 8.0. The significant change in Tm may be linked to dif- modynamic parameters of RNA thermal melting are shown ferent RNA conformations existing at near neutral and acidic in Figure 4B. According to DSC analysis, the melting temper- pH. It is consistent with the existence of neutral adenine bases ature is 55.6°C, 62.1°C, and 61.0°C at pH 3.5, 6.5, and 7.4, re- at near neutral or basic pH, which could destabilize the cen- spectively, which is in good agreement with Tm values tral wobble base pairs, inducing a different RNA structure. calculated by UV melting experiments. In particular, the Moreover, a higher Tm for the RNA at neutral pH indicates DSC analysis confirms that the RNA behaves similarly at

FIGURE 4. pH-dependent variation of physical and thermodynamic properties of RNA. (A) UV thermal melting curves at pH 3.5, 6.5, and 8.0 (or- ange, green, and blue) indicate a pH-dependent structural transition in RNA. (B) RNA thermal melting curves recorded with differential scanning calorimetry (DSC) at pH 3.5, 6.5, and 8.0.

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A novel RNA helix based on wobble base pairing

tion analyzed by primer extension) analysis (Wilkinson et al. TABLE 4. Thermodynamic parameters from DSC measurements of the RNA at pH values between 3.5 and 7.4 2006) for an oligonucleotide with a very similar sequence, r (UGUUCUCUACGAAGAACU), which also acquires a tri- Δ pH ± SD (°C) H ± SD (Kcal/mol) loop RNA stem–loop structure at physiological pH (Mino 3.5 55.66 ± 4.39 63.68 ± 1.63 et al. 2015). 6.5 62.15 ± 0.57 73.81 ± 9.93 7.4 61.01 ± 0.67 72.97 ± 1.27 DISCUSSION We present a 1.38-Å resolution crystal structure of an 18-nt- pH 6.5 and 7.4, whereas the melting transition at acidic pH long antiparallel RNA double helix formed in an acidic envi- occurs at a different temperature. The higher Tm of the ronment. The overall structure exhibits A-RNA conforma- RNA at neutral pH is in accordance with a higher energy re- tion and is clearly divided into three helical segments of 6 lease upon melting transition. The enthalpy change of RNA is ∼ ∼ bp each, denoted as WC, W, and WC helix. The W helix rep- 73 kcal/mol near physiological pH and 64 kcal/mol for the resents a unique and novel helical form of RNA that is double-helical structure existing at acidic pH, suggesting the completely based on wobble base pairing and consists of existence of different initial RNA structures (Table 4). Taken four C·A+ and two G·U wobble base pairs. The W helix together with the UV melting experiment, the DSC data con- exhibits overall helical similarity with A-RNA (Schindelin firm that different RNA structures exist at neutral and acidic et al. 1995; Olson et al. 2001), while it resembles A′-RNA pH for a sequence that permits formation of a central W helix in having 12 bp per turn (Arnott et al. 1972; Tanaka et al. with N1-protonation of adenine bases. 1999). In both G·U wobble pairs of the W-helical segment, guanine is shifted toward the minor groove and interacts ′ pH-induced structural changes probed by CD melting with O2 and O2 via a water molecule similar to isolated G·U pairs or tandem G·U pairs in motifs I and II of ribosomal Circular dichroism is extremely sensitive to nucleic- (Biswas and Sundaralingam 1997; Biswas et al. 1997). conformation and can provide valuable information about The C·A wobble pairs are formed as C·A+ pairs following its structure (Ivanov et al. 1973; Johnson 1990; Woody 1995). We probed the structural differences in RNA at different pH values by recording CD melting transitions at pH 3.5 and 7.5. Average CD spectra from three independent experiments for both pH values at 20°C and 90°C, respective- ly, representing the folded and melted state of RNA, are shown in Figure 5, while CD spectra and ellipticity changes with temperature are shown in Supplemental Figure S5. Clearly, at both pH values, the RNA exhibits CD spectra characteristic of the A conformation with maximum at ∼265 nm and minimum at ∼210 nm (Johnson 1990; Chauca-Diaz et al. 2015). Upon heating RNA to 90°C, bases become unstacked, which is reflected by disappearance of the peak at 210 nm, while the peak at ∼265 nm is reduced and shifted to ∼274 nm (Causley and Johnson 1982; Newbury et al. 1996). The peak at ∼210 nm is known to be related to the intrastrand interactions in RNA duplexes (Gray et al. 1981; Newbury et al. 1996), and our CD spectra show a significant increase in peak intensity at 210 nm at pH 7.5, suggesting extensive intrastrand interactions in the RNA. Moreover, we also observe a minor negative peak at ∼240 nm in the spectra, which is indicative of the presence of single-stranded RNA at pH 7.5 (Newbury et al. 1996; Chauca-Diaz et al. 2015). All CD observations are in agreement with RNA acquiring a highly stable stem–loop structure with unprotonated ade- FIGURE 5. Structural asymmetries of RNA element at different pH are nines at physiological pH, forming extensive intrastrand in- probed by CD melting spectra. (A) An overlay of CD spectra at 20°C teractions as compared to the double-helical structure, and (solid line) and 90°C (dotted line) for RNA elements is represented at pH 3.5 (black) and 7.5 (blue). (B) The physiological structure of the unpaired bases in the loop (see Fig. 5). Our CD data are RNA element is most likely an A-RNA stem–loop with either tri- or ′ strongly supported by a SHAPE (selective 2 -hydroxyl acyla- -loop and not the double-helical structure observed at acidic pH.

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N1 protonation of adenines in the W helix, which is indicated occurs under acidic conditions and seems to be nonphysio- by difference electron density and adenine bond angles after logical. Previous reports, however, suggest that microscopic unrestrained structure refinement that agree with bond-angle pKa change could lead to formation of C·A+ pairs in cells standards from adenine bases with N1 protonation (Taylor and influence RNA cleavage by leadzymes (Legault and and Kennard 1982; Safaee et al. 2013; Gleghorn et al. 2016). Pardi 1994, 1997) and a hairpin (Cai and Tinoco C·A+ base pairs in previous RNA crystal structures, either in 1996). A C·A+ pair can also substitute for G·U in the P1 helix isolation (Pan et al. 1998) or in tandem (Jang et al. 1998), in group I introns. We suggest that similar structural changes resulted in RNA bending of ∼20°. The present structure of a in the RNA element studied here might occur in cells, as a re- W-helical segment with flanking A-RNA does not show any sult of microscopic pKa change, in order to regulate the ex- significant bending at the base-pair steps involving C·A+,re- pression of downstream immune response factors by sulting in a straight helical half turn of RNA. endoribonuclease Regnase1. However, no such report on An RNA structure with tandem C·A+ pairs in a CA+ RNA structural change has been published so far. dinucleotide step exhibited cross-strand purine stacking (Supplemental Fig. S6A) accompanied by helical over-wind- ing toward the neighboring Watson–Crick pairs (Jang et al. MATERIALS AND METHODS 1998), while isolated C·A+ pairs were incorporated in A- form RNA without much helical perturbations (Hunter Oligonucleotide crystallization et al. 1987; Pan et al. 1998). In the presented structure, + + The HPLC-purified oligonucleotide r(UGUUCUCUACGAAGAACA) however, tandem C·A pairs in an A C dinucleotide step corresponding to a highly conserved stem–loop region in the 3′ UTR are stacked almost parallel (Supplemental Fig. S6B) with of human interleukin (IL)-6 mRNA was purchased from Eurofins under-winding of neighboring pairs. Although helical pa- (Berlin, Germany) and dissolved in 20 mM Tris (pH rameters, in particular the base pair Twist, in the central W 7.0), 50 mM KCl to 1 mM concentration. RNA was reannealed segment show strong fluctuations, their mean values remain by heating at 90°C for 10 min followed by snap-cooling on ice for in the A-helix regime. Overall, we present a novel RNA struc- 5 min and then diluted in suitable reaction buffer before experi- ture, the W helix, in which six consecutive wobble base pairs ments. Crystals were obtained during co-crystallization trials of are incorporated in standard A-form RNA, extending the RNA oligonucleotide and a catalytically inactive mutant of the range of RNA conformations observed until now. mouse ZC3H12C mixed in a 2:1 molar ratio. A total – Importantly, we observed that the studied oligoribonu- of 0.2 µL of protein RNA complex in 25 mM HEPES (pH 7.5), 150 mM NaCl, 20 µM ZnSO , 0.5 mM MgCl was mixed with cleotide exhibits significant differences in thermal stability 4 2 0.2 µL of 20% PEG 6000, 1.0 M LiCl, 0.1 M citric acid (pH 4.0) at different pH values as probed by UV and DSC thermal at 4°C using sitting-drop vapor diffusion technique, and rod-shaped melting experiments. In both UV melting and DSC analysis, crystals were harvested after ∼75 d, soaked in reservoir solution with we found that the thermodynamic properties of RNA varied 20% ethylene glycol before flash freezing in liquid nitrogen. Mixing between acidic pH of 3.5 and pH 6.5, which is consistent with equal volumes of solutions of the protein–RNA complex and the adenine-N1 protonation at acidic pH (Saenger 1983; Kapinos crystallization buffer as above yields a solution with a pH between et al. 2011). We conclude that the observed differences in the 4.4 and 4.5. melting behavior result from adenine protonation at low pH. The adenine protonation state remains unchanged between pH 6.5 and 8.0, therefore we observe no differences in ther- X-ray data collection, structure determination, modynamic properties of RNA in this pH regime. Moreover, and refinement the significant difference in the enthalpic contribution to the X-ray diffraction data were collected at beamline 14.1 of the BESSY II melting transition suggests that unfolding follows different synchrotron operated by Helmholtz-Zentrum Berlin (Mueller et al. routes at different pH, consistent with the existence of differ- 2012) at a wavelength of 0.9184 Å. Initial indexing and data collec- ent initial RNA structures. Comparative analyses of CD tion strategy were determined by iMOSFLM (Battye et al. 2011), spectra at different pH values point to the existence of either and a complete X-ray data set was processed with XDSAPP (Krug a tri-loop (Mino et al. 2015) or a hexa-loop (Zuker 2003) et al. 2012). Since we expected the RNA to form a stem–loop struc- RNA stem–loop structure, which acquires a double-helical ture, we used the 5 bp stem of pre-let-7f-1 RNA (PDB entry 3TS0) wobble-base-paired RNA structure due to protonation of as a search model (Nam et al. 2011) for molecular replacement using the central adenines at low pH. the PHASER-MR program (McCoy et al. 2005). Autobuilding of the As we have demonstrated that the RNA oligonucleotide ex- initial model was performed with Phenix (Adams et al. 2010). Canonical hydrogens were generated by Phenix.reduce (Word et al. ists in two stable conformations at different pH values, we 1999) and included in the refinement as riding hydrogens. The propose that this molecule, as well as similar RNA elements graphics program WinCoot (Emsley et al. 2010) was used for model with a propensity for forming W-helical structures with building and visualization, and all refinements were performed by + C·A base pairs, may be useful tools as pH biosensors and Phenix.Refine or CCP4 Refmac5 (Murshudov et al. 1997). Molecular in RNA nanotechnology applications (Yan 2004; Guo 2010; drawings were generated with the PyMOL molecular graphics system Yu et al. 2015; Amodio et al. 2016). Adenine-N1 protonation (Version 1.7.0.5, Schrodinger, LLC), and Web 3DNA (Zheng et al.

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A novel RNA helix based on wobble base pairing

2009) was used to analyze the structure. Atomic coordinates and Received September 15, 2017; accepted November 5, 2017. structure factors for the RNA double helix were deposited in the Pro- tein Data Bank under accession number 5NXT. REFERENCES UV thermal denaturation Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. 2010. RNA UV thermal melting experiments were performed at three dif- PHENIX: a comprehensive Python-based system for macromolecu- ferent pH values in reaction buffers (i) 25 mM citric acid (pH 3.5), lar structure solution. Acta Crystallogr D Biol Crystallogr 66: 213–221. 200 mM LiCl, 1 mM MgSO4, (ii) 25 mM citric acid (pH 6.5), – Amodio A, Adedeji AF, Castronovo M, Franco , Ricci . 2016. pH- 200 mM LiCl, 1 mM MgSO4, or (iii) 25 mM Tris HCl (pH 8.0), controlled assembly of DNA tiles. 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A novel form of RNA double helix based on G·U and C·A+ wobble base pairing

Ankur Garg and Udo Heinemann

RNA 2018 24: 209-218 originally published online November 9, 2017 Access the most recent version at doi:10.1261/.064048.117

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